Process for separation of polynucleotide fragments

ABSTRACT

A batch process for obtaining polynucleotide fragments, such as dsDNA, having a selected size from a mixture of polynucleotide fragments including the steps of a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface; b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium. The binding medium can be organic polymer or inorganic particle beads. The mixture of polynucleotides can be the product of a PCR amplification. The binding medium can be contained within a column, a web or a container.

CROSS REFERENCE TO RELATED COPENDING APPLICATIONS

[0001] This application is a regular U.S. patent application under 35U.S.C. §111(a) and claims priority from the copending, commonly assignedU.S. provisional application No. 60/044,856, filed Apr. 25, 1997, under35 U.S.C. §111(b).

FIELD OF THE INVENTION

[0002] The invention pertains to the field of isolation and purificationof polynucleotides. In particular, the invention relates to a processfor purification of polynucleotides.

BACKGROUND OF THE INVENTION

[0003] The separation and quantification of polynucleotides such as DNAis of critical importance in molecular biology and improved methods area focus of current interest. One separation method includessize-exclusion chromatography (E. Heftmann, in J. Chromatog. Lib., Vol.51A, p. A299 (1992)). The disadvantages of this method include lowresolution and low capacity. Another separation method is anion exchangechromatography of DNA with tetramethylammonium chloride containingmobile phases as described in European patent application 0 507 591 A2to Bloch. However, the separation is not strictly size-based, and theresolution is not always adequate. A further disadvantage of methodswhich rely on binding of anionic DNA includes the required use of highconcentrations of nonvolatile salts in the mobile phase; this interfereswith subsequent isolation and measurement (e.g. mass spectral analysis)on the separated fragments.

[0004] Thus there is a need in the art for a size-based separationprocess for DNA which has high capacity and resolution, and which doesnot require use of nonvolatile salts.

SUMMARY OF THE INVENTION

[0005] Briefly, the instant invention comprises a process fornon-specifically binding all of the fragments in a polynucleotidemixture onto a solid binding medium having a hydrophobic surface in thepresence of a counterion agent, and the selective release of thefragments based on their size, from smallest to largest, as theconcentration of the organic component of the mobile phase is increased.In one embodiment, the binding medium is comprised of beads having ahydrophobic surface. The process does not require use of a high pressureliquid chromatography (HPLC) system and is amenable to scale up orminiaturization.

[0006] Accordingly, one aspect of the present invention provides aprocess for separating a mixture of polynucleotides which is based onthe base-pair length of the fragments.

[0007] As another aspect of the present invention, there is provided aseparation process for a mixture of polynucleotides which can be carriedout using binding medium incorporated into a variety of separationconfigurations including a container such as a column or well.

[0008] In another aspect, the present invention provides a separationprocess for a mixture of polynucleotides which utilizes a hydrophobicbinding medium enmeshed in an inert fiber matrix.

[0009] In yet another aspect, the present invention provides a simple,inexpensive, and rapid process, for separating a mixture ofpolynucleotide fragments.

[0010] In a further aspect, the present invention provides a separationprocess for polynucleotide fragments which does not require the use ofnonvolatile salts.

[0011] One embodiment of the instant invention is directed to a batchprocess for obtaining polynucleotide fragments (such as dsDNApolynucleotides) having a selected size from a mixture of polynucleotidefragments, comprising the steps of

[0012] (a) applying a solution of the mixture of polynucleotidefragments and a counterion agent to a binding medium having ahydrophobic surface;

[0013] (b) contacting the binding medium with a first stripping solventand counterion agent, the first stripping solvent having a concentrationof organic component, such as acetonitrile, sufficient to release fromthe binding medium all polynucleotide fragments having a size smallerthan the selected size, and removing the first stripping solvent fromthe binding medium; and

[0014] (c) contacting the binding medium with a second stripping solventhaving a concentration of organic component sufficient to release fromthe binding medium the polynucleotide fragments having the selectedsize, and removing the second stripping solvent from the binding medium.

[0015] The binding medium can be rinsed with fresh first strippingsolvent following step (b) to remove residual fragments having a sizesmaller than the selected size therefrom. The binding medium can also berinsed with fresh second stripping solvent following step (c) to removeresidual polynucleotide fragments of the selected size therefrom. Thecounterion agent preferably is triethylammonium acetate ortriethylammonium hexafluoroisopropyl alcohol. The binding medium ispreferably porous or nonporous beads having a diameter of from about 1.0to 1,000 μm. The beads can consist of organic polymer such as acopolymer of vinyl aromatic monomers selected from the group consistingof styrene, alkyl substituted styrene, alpha-methylstyrene and alphasubstituted alpha-methylstyrene. Alternatively, the beads can compriseinorganic particles such as silica, silica carbide, silica nitrite,titanium oxide, aluminum oxide, zirconium oxide modified to have ahydrophobic surface. The hydrophobic surface can be an organic polymersupported on the inorganic particle. The hydrophobic surface can be longchain hydrocarbons having from 8-24 carbons bonded to the inorganicparticle. Preferably, any residual polar groups of the inorganicparticle have been end-capped with trimethylsilyl chloride orhexamethyidisilazane.

[0016] The process of the invention is particulary useful in separatingthe products of a PCR amplification.

[0017] The binding medium can be contained within a column, a web or acontainer. In one embodiment, the medium (such as beads) is containedwithin a web consisting of an inert fiber matrix and beads enmeshed inthe matrix. In a preferred embodiment, the binding medium consists ofhydrophobic beads which are contained a polymeric (such aspolytetrafluoroethylene) fibril matrix with the ratio of beads to fibrilmatrix being in the range of 29:1 to 4:1 by weight.

[0018] Other aspects and advantages of the present invention aredescribed further in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a separation of pUC18-DNA HaeIII digest on a columnpacked with a nonporous poly(styrene-divinylbenzene) polymer beads. Thecolumn dimensions were 30 mm×4.6 mm i.d.

[0020]FIG. 2 is a separation of pUC18-DNA HaeIII digest on a columnpacked with a nonporous poly(styrene-divinylbenzene) polymer beads. Thecolumn dimensions were 50 mm×4.6 mm i.d.

[0021]FIG. 3 is a separation of pUC18-DNA HaeIII digest on a columnpacked with a nonporous poly(styrene-divinylbenzene) polymer beads. Thecolumn dimensions were 50 mm×6.5 mm i.d.

[0022]FIG. 4 is a separation of pUC18-DNA HaeIII digest (20 times moresample was injected as compared to FIG. 3) on a column packed with anonporous poly(styrene-divinylbenzene) polymer beads. The columndimensions were 50 mm×6.5 mm i.d.

[0023]FIG. 5 is a separation of pUC18-DNA HaeIII digest on two discscontaining binding media placed in series and containing nonporouspoly(styrene-divinylbenzene) polymer beads. The dimensions of each discwas 0.7 mm×4.6 mm i.d.

[0024]FIG. 6 shows the release of eight DNA fragments from polymer beadsin single equilibria bulk separations (under conditions as described inTABLE 1) showing the dependence on the acetonitrile concentration.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The term “polynucleotide” is defined as a linear polymercontaining an indefinite number of nucleotides, linked from one ribose(or deoxyribose) to another via phosphoric residues. The presentinvention can be used in the separation of RNA or of double- orsingle-stranded DNA or of synthetic DNA analogs. The polynucleotide canbe a linear molecule or a closed circle and can be modified, e.g.labeled with biotin or fluorescent molecules. For purposes ofsimplifying the description of the invention, and not by way oflimitation, the separation of double-stranded DNA will be described inthe examples herein, it being understood that all polynucleotides areintended to be included within the scope of this invention.

[0026] A “counterion agent” is a compound used to form a neutral saltwith anionic DNA. Counterion agents that are volatile, such astrialkylammonium acetate and trialkylammonium carbonate, are preferredfor use in the method of the invention, with triethylammonium acetate(TEAA) and triethylammonium hexafluoroisopropyl alcohol being mostpreferred.

[0027] “Non-specific binding” refers to binding to all of the DNAfragments in a mixture despite differences in the DNA sequence or sizeof the different DNA molecules. In the present invention, such bindingoccurs when the fragments are exposed to the hydrophobic surface of abinding medium in a stripping solvent containing a counterion agent butlacking organic component or at low concentrations of organic component.

[0028] “Binding medium” refers to a solid phase having a hydrophobicsurface suitable for binding DNA fragments in the presence of an aqueousphase containing a counterion agent. Examples include beads orparticles.

[0029] “Organic component” refers to a water-soluble organic solventwhich is suitable for use in an aqueous solution in the presentinvention for releasing polynucleotides from the hydrophobic surface ofthe binding medium.

[0030] “Stripping solvent” refers to an aqueous solution containing anorganic component for use in the present invention for releasingpolynucleotides from the hydrophobic surface of the binding medium.

[0031] “Organic component” refers to a water-soluble organic solventwhich is suitable for use in the stripping solvent of the presentinvention.

[0032] As demonstrated by Bonn et al. in U.S. Pat. No. 6,585,236, use ofhydrophobic beads in HPLC gave size-based separation of polynucleotides.In the presence of a counterion agent, polynucleotides eluted in theorder of smallest to largest as the concentration of the organiccomponent of the mobile phase was increased.

[0033] In traditional reverse-phase HPLC separations, variations in thelength of the column alter the elution profile of the analyte due topartitioning of the analyte between the stationary phase and the mobilephase. As will be demonstrated hereinbelow, applicants have discoveredthat when hydrophobic beads were used as the separation medium of a HPLCcolumn, no change in the elution profile of polynucleotide fragments wasobserved with a change in the length of the separation column.Applicants have discovered that size-based separation of polynucleotidescan also be achieved using a variety of hydrophobic binding mediawithout using HPLC systems. This makes possible processes forseparations of polynucleotides not heretofore known in the art.

[0034] In U.S. Pat. No. 5,585,236, Bonn et al. had characterized thepolynucleotide separation process as reverse phase ion pairingchromatography (RPIPC). However, since RPIPC does not incorporatecertain essential characteristics described in the present invention,another term, Matched Ion Polynucleotide Chromatography (MIPC), has beenselected. MIPC as used herein, is defined as a process for separatingsingle and double stranded polynucleotides using non-polar beads,wherein the process uses a counterion agent, and a stripping solvent torelease selected polynucleotide fragments from the beads.

[0035] The present invention is a process for binding DNAnon-specifically and reversibly in the presence of a counterion to asolid phase binding medium, such as beads, having a hydrophobic surface.In the process of the invention, the DNA can be present in solution withwater or in a reaction buffer, Such a solution can also contain othercomponents, such as other biomolecules, inorganic compounds and organiccompounds as long as such other components do not interferesignificantly with the binding process of the invention. As an example,the solution can be a reaction product of a PCR amplification, and theprocess of the present invention can be used to separate impurities,such as primers or primer dimers, from the amplified sequence.

[0036] The present invention requires a counterion agent for forming ahydrophobic salt with anionic DNA to enable the hydrophobic interactionof the DNA-counterion with the binding medium. Counterion agents thatare volatile, such as trialkylammonium acetate and trialkylammoniumcarbonate, are preferred for use in the process of the invention, withtriethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropylalcohol being most preferred. Trialkylammonium phosphate can also beused. The counterion agent can be added to the DNA mixture first or theDNA mixture can be injected into a polar stripping solvent containingthe counterion agent. Preferred counterion agents are those which areeasily removed after the separation process.

[0037] The present invention utilizes an aqueous stripping solventcontaining organic component. At increasing concentrations of theorganic component in the stripping solvent, DNA fragments can bereleased from the binding medium as a function of the size of thefragments, from smallest to largest. Preferred stripping solvents mustbe able to both release the DNA-counterion from the surface and maintainthe DNA-counterion in solution. Preferred stripping solvents do notinterfere with the isolation or recovery of the fragments and are easilyremoved after the separation. The solvent is preferably selected fromthe group consisting of alcohols, nitrites, dimethylformamide, esters,and ethers. Examples of suitable solvents include acetonitrile,alcohols, 2-propanol, methanol, tetrahydrofuran, and 1,4 dioxane. Theconcentration of organic component and counterion agent required for aseparation will depend on the binding medium used, and the conditionsare optimized by routine methods.

[0038] In the process of the present invention, the release of thefragments from the surface can be modulated by exposing the surface ofthe binding medium to variations in parameters such as temperature andpH. The release of fragments can also be modulated by chemicalinteractions, such as the use of an additive, e.g. a second, more polarcounterion agent, in the stripping solvent that would competitively forma stable complex with the DNA-counterion fragments, and would be able torelease the fragments from the surface of the medium,

[0039] The fragments in solution can be detected by any suitable methodsuch as by UV absorbance or other means (e.g. fluorescence,radioactivity).

[0040] The process of the present invention requires a solid phasebinding medium having a suitable surface for binding the DNA-counterion.In a preferred embodiment, the medium comprises separation beads orparticles. A wide variety of surface chemistries can be used in thepresent invention, but the preferred bead surface is hydrophobic. Thepreferred surface does not contribute to competing mechanisms, ex. anionexchange or size-exclusion, and preferably does not hinder binding orrelease of the DNA-counterion by steric or energetic interferingfactors.

[0041] Examples of suitable beads for use in the present inventioninclude polymer beads as described in copending U.S. patent applicationSer. No. 09/058,580, filed on Apr. 10, 1998, and coated inorganicparticles as described in copending U.S. patent application Ser. No.09/058,337, filed on Apr. 10, 1998.

[0042] Chromatographic efficiency of the beads is predominantlyinfluenced by the properties of surface and near-surface areas. The mainbody or the center of such beads can exhibit entirely differentchemistries and physical properties from those observed at or near thesurface of the beads.

[0043] An example of a suitable bead is a porous or nonporous polymerbead comprised of a copolymer of vinyl aromatic monomers. Examples ofvinyl aromatic monomers include styrene, alkyl substituted styrene,alpha-methylstyrene, and alkyl substituted alpha-methylstyrene.

[0044] Another example of a suitable bead is a porous or nonporousparticle such as silica, silica carbide, silica nitrite, titanium oxide,aluminum oxide, zirconium oxide which is modified to have a hydrophobicsurface. The hydrophobic surface can be an organic polymer supported onthe inorganic particle. In one embodiment, the hydrophobic surfaceincludes long chain hydrocarbons having from 8-24 carbons bonded to theinorganic oxide particle. An example is a silica particle havingsubstantially all surface substrate groups reacted with a hydrocarbongroup and then endcapped with a non-polar hydrocarbon or substitutedhydrocarbon group, preferably a tri(lower alkyl)chlorosilane ortetra(lower alkyl)dichlorodisilazane. The particle can be end-cappedwith trimethylsilyl chloride or hexamethyidisilazane.

[0045] Beads useful in the present process can be a variety of shapes,which can be regular or irregular; preferably the shape maximizes thesurface area of the beads. The beads should be of such a size that theirseparation from solution, for example by filtration or centrifugation,is not difficult.

[0046] In one embodiment, the beads of the present invention have adiameter of about 1 to 100 μm and can be used in an HPLC system. Intraditional reverse-phase HPLC separations, variation in the length ofthe column alters the elution profile of the analyte due to partitioningof the analyte between the stationary phase and the stripping solvent.For a given sample, differences in selectivity and resolution will beobserved when the column length is changed. It has now been surprisinglydiscovered by applicants that this is not the case for DNA separationson hydrophobic beads as described herein and this observation is thebasis for the process of the present invention. This phenomenon isillustrated by the separations described in FIGS. 1 and 2. Referring toFIG. 1, a DNA mixture was separated as described in EXAMPLE 1. It can beseen that the separation profiles were very similar. Shorter columns canbe used without diminishing the resolution, which would have theadvantages of lower back-pressure, good flow paths with small extracolumn effects. In addition, peak broadening would be reduced in shortercolumns due to less diffusion of the solutes. It was also observed thatthe capacity of the column could be increased by increasing the diameterof the column without a decrease in separation performance, as shown inFIGS. 2 and 3. In FIG. 3, the separation conditions were the same as forFIG. 2 except that a column having a wider bore was used.

[0047] As described in EXAMPLE 2, in an experiment to assess thecapacity of the nonporous polymer beads, FIG. 4 shows a separation usingthe same conditions as for FIG. 3, but after injecting a sample havingabout 20-fold more DNA. The increase in sample loading did not have asignificant effect on the separation performance. The capacity of thebeads used in EXAMPLES 1 and 2 was about 10 μg DNA per gram of beads.

[0048] Referring to FIG. 5 and EXAMPLE 4, a DNA fragment separation wasperformed using discs of 0.7 mm thickness, and demonstrated thatseparation is possible using a thin separation bed containinghydrophobic binding medium. The resolution is adequate for someapplications such as cleanup of the product of a PCR amplification.

[0049] An example would be the separation of a PCR product from residualprimers, dNTP material, and primer dimers. Primer dimers form when thetwo primers associate with a 2 to 3 base pair overlap and are often abyproduct of the PCR reaction. These dimers form when the two primersassociate, e.g. with a 2 to 3 base pair overlap. These dimers areapproximately the size of the two primers less 1 to 3 base pairs. Sinceprimer dimers are double stranded, they are particularly difficult toremove from PCR products, but are readily resolved using the process ofthe present invention as demonstrated in EXAMPLE 5.

[0050] The geometry and configuration of the container supporting thebinding medium can be varied without loss of the ability to separate DNAfragments by size. For example, purification of large-scale DNA mixturesis possible, and could be used as a preparative step in obtainingpharmaceutical grade polynucleotides.

[0051] In one embodiment of the process of the present invention, theseparation can be conducted as a batch process in a container. Thevolume of the container can vary widely depending on the amount ofmixture to be separated. The container can be a column, a flask, a well,or a tank, for example. The size of such a container can be as small asa well on a 96-well microtiter plate or as large as a multi-liter vat,for example. The binding medium can be beads. Beads useful in the batchprocess can be a variety of shapes, which can be regular or irregular;preferably the shape maximizes the surface area of the beads. The beadsshould be of such a size that their separation from solution, forexample by filtration or centrifugation, is not difficult.

[0052] In one example, the DNA mixture, counterion, and beads are mixedin bulk with a polar solution in a container, and binding is allowed tooccur. Preferably, all of the DNA-counterion will bind nonspecificallyto the beads under the initial conditions in which the stripping solventhas low concentration of organic component. To release DNA fragmentsfrom the beads, the beads are brought into contact with a strippingsolvent of sufficient concentration of organic component. Elutionconditions for DNA fragments having a selected base-pair length can bepredetermined, e.g. by determining the elution profile of a standard DNAmixture at various concentrations of stripping solvent. This calibrationprocedure can be conducted on a small scale and applied to a large-scaleprocess. Specific stripping solvent compositions can be adjusted toelute polynucleotide fragments of any specific base pair size. Anexample of the high resolution which can be obtained in a singleequilibria bulk process is exemplified by referring to FIG. 6 andEXAMPLE 3 where isolation of a 102 base pair fragment was achieved byincrementally increasing the ACN concentration from 14.6% to 15.9%.

[0053] In a preferred embodiment of the process of the invention, afterthe sample mixture is bound, stripping solvent is applied in a firstrelease step in which the organic component is applied at aconcentration which will release fragments smaller than the desiredfragment. The beads are then separated from the stripping solvent, e.g.by centrifugation or by filtration. Stripping solvent is then applied tothe beads in a second release step in which the stripping solvent isapplied at an elevated concentration, e.g. an incrementally elevatedconcentration, which selectively releases the desired size of fragment.Optionally, the process can be repeated with stripping solvent appliedat increasing concentrations of organic component in order to releaselonger fragments of discrete base pair length. Each fragment can berecovered, e.g. by collecting the stripping solvent at eachconcentration of organic component. It is possible to have multiple washsteps at a single concentration of stripping solvent to ensure completeremoval of the desired size fragment.

[0054] In another example of a batch process of the present invention,the separation is performed using a column, e.g. an open column undergravity flow conditions or a low pressure column equipped with aperistaltic pump. The binding medium comprises beads having a diameterlarge enough to permit flow of stripping solvent without requiring highpressure pumps. Preferred beads have a diameter of about 20 to 1000microns and can be made from various materials as described hereinabove.The dimensions of the column can range from about 10 cm to 1 m inlength, and 1 to 100 cm in diameter, for example. In operation, thecolumn is first conditioned using a polar solvent. A DNA-counterionmixture is applied to the column in a convenient volume such as from 1to 50 mL. For dilute samples having a large volume, the sample can beapplied continuously, or in stages, to “load” the column. Preferably,all of the DNA-counterion will bind to the binding medium under theinitial conditions in which the stripping solvent has low concentrationof organic component. To release DNA fragments from the separationbeads, the beads are brought into contact with a stripping solvent ofsufficient concentration of organic component. Elution conditions for aDNA fragment having a selected base-pair length can be predetermined,e.g. by determining the elution profile of a standard DNA mixture atvarious concentrations of stripping solvent. This calibration procedurecan be conducted on a small scale and applied to a large-scale process.Specific stripping solvent compositions can be adjusted to elutepolynucleotide fragments of any specific base-pair size in analogy tothe bulk equilibria process as described hereinabove. After the samplemixture is bound to the binding medium in the column, stripping solventis applied in a first release step in which the organic component ispresent at a concentration which will release fragments smaller than thedesired fragment; stripping solvent is then applied in a second releasestep in which the organic component is present at an elevatedconcentration, e.g. an incrementally elevated concentration, whichselectively releases the desired fragment. Optionally, stripping solventcan be applied in a gradient of increasing concentration of organiccomponent, e.g. a step-gradient or continuous gradient, in order torelease longer fragments. By using a step gradient of increasing ACNconcentration, larger fragments can be removed in discrete base pairlengths from the separation beads and isolated. Each fragment isrecovered, for example, by collecting the stripping solvent at eachconcentration of organic componennt. For each fragment, the separationprocess can be repeated, if necessary, e.g. by application to anothercolumn.

[0055] In another embodiment of the invention, the binding medium can beretained in a web or pad. An example is a web of inert fiber matrix withhydrophobic binding medium, such as the beads as described hereinabove,enmeshed in the matrix. The web of the present invention is a compositearticle comprising binding medium which has been incorporated into afabric or membrane. The term “incorporated into a fabric membrane” meansthat the binding medium is encapsulated by or trapped within a fabric ormembrane, is stabilized within a fabric or membrane or is covalentlyattached to a fabric or membrane such that the binding medium does notexist as free flowable particulate bulk material and is not separablefrom the web under liquid chromatography conditions.

[0056] When the binding material is incorporated into a web, the web maybe woven or non-woven. The spaces between fibers of the web should besmall enough to prevent binding medium material from passing through theweb. The density of non-woven fibers and the density of warp and weftfibers of the web can be routinely adjusted to provide the desireddensity and porosity.

[0057] The web fibers can be made of any suitable material so long asthe material is porous. Suitable materials are described in U.S. Pat.No. 5,338,448 to Gjerde. Generally, the fibers will be made of a poroussynthetic or natural polymeric material, e.g. polytetrafluoroethylene,cellulose, polyvinyl chloride, nylon, etc. The DNA in the samplepreferably binds only to the binding medium and the binding is notdetrimentally affected by the fiber matrix material. When the bindingmedium consists of polymeric beads, the ratio of beads to fiber matrixmaterial can be in the range of 19:1 to 4:1 by weight, for example.

[0058] In one embodiment, the web is mounted on a support and the sampleis applied and eluted in a manner analogous to the open column processas described hereinabove. The web material can be packed into a column.An advantage of using a web material is that it provides flexibility inhow thin a column bed can be made, e.g. the web can be formed as a disk.Also, several uniform beds can be made at once. Multiple webs can besupported in a row or adapted to a matrix well format, e.g. a 4, 8, 16,or 96 well plate. The web can be used in analogy to the bulk equilibriaprocess or column as described hereinabove with a binding step followedby release steps.

[0059] An example of a suitable fibril matrix is polytetrafluoroethylene(PTFE) as described in U.S. Pat. No. 4,906,378 to Hagen. The ratio ofbeads to PTFE fibril matrix can be in the range of 19:1 to 4:1 byweight, for example.

[0060] The process of the invention preferably includes precautions toprevent contamination with multivalent cations such as Fe(III), Cr(III),or colloidal metal contaminants. Multivalent cations can causenon-specific binding of the DNA to the surfaces of conduits andcontainers which can lead to low recovery. The inner surfaces, whichcontact liquids within the system, preferably are treated to removemultivalent contaminants, e.g. treating with an acid such as nitricacid. The efficiency of the separation process may be enhanced by theoptional addition of a chelating agent such as EDTA, e.g. at aconcentration of 0 to 0.1 M. Suitable precautions are described incopending U.S. patent application Ser. No. 08/748,376 filed Nov. 13,1996. Precautions can also be taken during the manufacture of thebinding medium to prevent contamination with multivalent cations.Examples of suitable precautions in the manufacture of beads, forexample, are described in copending U.S. patent application Ser. No.09/058,580, filed on Apr. 10, 1998, and in copending U.S. patentapplication Ser. No. 09/058,337, filed on Apr. 10, 1998.

[0061] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

[0062] All references cited herein are hereby incorporated by referencein their entirety.

[0063] Procedures described in the past tense in the examples below havebeen carried out in the laboratory. Procedures described in the presenttense have not been carried out in the laboratory, and areconstructively reduced to practice with the filing of this application.

EXAMPLE 1 Effect of Column Dimensions on the HPLC Separation of DNAFragments

[0064] In the separation shown in FIG. 1, the HPLC column was packedwith 2.1 micron C-18 alkylated nonporous poly(styrene-divinylbenzene)polymer beads and a DNA separation was run under the followingconditions:

[0065] Column: 30×4.6 mm i.d.; stripping solvent 0.1 M TEAA, pH 7.2;gradient: 35-55% acetonitrile (ACN) in 3 min, 55-65% ACN in 7 min, 65%ACN for 2.5 min; 100% ACN for 1.5 min, back to 35% ACN in 2 min. Theflow rate was 0.75 mL/min, p=1300 psi, detection UV at 260 nm, columntemp. 51° C. The sample was 3 μL (=0.12 μg pUC18 DNA-HaeIII digest). Forcomparison, FIG. 2 shows a separation using a longer column packed withthe same sized beads under similar elution conditions: Column: 50×4.6 mmi.d.; stripping solvent 0.1 M TEAA, pH 7.2; gradient: 35-55% ACN in 3min, 55-65% ACN in 7 min, 65% ACN for 2.5 min; 100% ACN for 1.5 min,back to 35% ACN in 2 min. The flow rate was 0.75 mL/min, p=1650 psi,detection UV at 260 nm, column temp. 51° C. The sample was 5 μL (=0.2 μgpUC18 DNA-HaeIII digest). In FIG. 3, the separation conditions weresimilar to those of FIG. 2 except that a column having a wider borediameter was used: the column was 50 mm×6.5 mm i.d. column, temp.=50°C., p=750 psi. The sample was 5 μL (=0.2 μg pUC18 DNA-HaeIII digest).

EXAMPLE 2 Effect of Sample Size on HPLC Separation of DNA Fragments

[0066] In an experiment to assess the effect of sample size on theseparation, FIG. 4 shows a separation using the same conditions as forFIG. 3, but after injecting a sample having about 20-fold more DNA (4.1μg pUC18 DNA-HaeIII digest in 10 μL). The increase in sample loading didnot significantly affect the separation performance.

EXAMPLE3 Separation of DNA Fragments Using a Single Equilibria BulkProcess

[0067] The separation of dsDNA fragments from a pUC18-DNA HaeIII digestwas performed using the same beads as used in EXAMPLES 1 and 2. Ninedifferent vials each containing 0.035 g of beads and 10 μL of DNA digest(4.5 μg) were mixed with 100 μL of 0.1 M triethylammonium acetate(TEAA), each vial containing different amounts of ACN. The incubationtime was 10 min at 23° C. The vials were centrifuged with a Brinkmanmodel 3200 table-top centrifuge for 5 minutes. A 3 μL aliquot of thesupernatant was removed by syringe for analysis. The analysis was doneusing HPLC with on-line UV detection at 260 nm. TABLE 1 shows theconcentration of DNA, TEAA, ACN and the amount of resin in the differentexperiments. TABLE I Amount of resin Volume DNA Exp. (g) (μL) % ACN TEAA(μg/μL) 1 0.035 110  7.88% 0.1M 0.0405 2 0.035 110  9.01% 0.1M 0.0405 30.035 110 10.14% 0.1M 0.0405 4 0.035 110 11.26% 0.1M 0.0405 5 0.035 11012.39% 0.1M 0.0405 6 0.035 110 13.51% 0.1M 0.0405 7 0.035 110 14.64%0.1M 0.0405 8 0.035 110 15.91% 0.1M 0.0405 9 0.035 110 17.05% 0.1M0.0405

[0068] Referring to FIG. 6, the experiments showed that the smallerfragments (80, 102, 174 bp) in this particular digest were releasedquantitatively from the resin surface by increasing the ACNconcentration from 15 to 16% (in solution) and the larger fragments(257, 267, 298, 434, and 587 bp) by increasing the ACN concentrationfrom 16 to 18.5%. Quantitative release for the 102 bp fragment wasachieved by increasing the ACN concentration from 14.6% to 15.9%.

EXAMPLE 4 Separation of DNA Fragments Using Discs

[0069]FIG. 5 shows the separation of dsDNA fragments from a pUC18-DNAHaeIII digest performed using 8 micron C-18 nonporouspoly(styrene-divinylbenzene) polymer beads in two discs placed inseries. The discs are available commercially under the trademark GuardDisc™ (Transgenomic, Inc., San Jose, Calif.) which contain beadsenmeshed in a web of TEFLON™ fibril matrix at a weight ratio of 9:1beads to fibril matrix.

[0070] The DNA separation was run under the following conditions: GuardDisc™ 0.7×4.6 mm i.d.; stripping solvent 0.1 M TEAA, pH 7.2; gradient:35-55% acetonitrile (ACN) in 3 min, 55-65% ACN in 7 min, 65% ACN for 2.5min; 100% ACN for 1.5 min, back to 35% ACN in 2 min. The flow rate was0.75 mL/min, detection UV at 260 nm, column temp. 51° C., p=50 psi. Thesample was 3 μL (=0.12 g pUC18 DNA-HaeIII digest).

EXAMPLE 5 Separation of PCR Reaction Products Using Discs

[0071] The reaction products of a PCR preparation are separated underthe conditions as described in EXAMPLE 4. Primer dimers elute in about2-3 minutes and are well resolved from a 405 base pair PCR product whichelutes in about 4-5 minutes.

[0072] While the foregoing has presented specific embodiments of thepresent invention, it is to be understood that these embodiments havebeen presented by way of example only. It is expected that others willperceive and practice variations which, though differing from theforegoing, do not depart from the spirit and scope of the invention asdescribed and claimed herein.

What is claimed is:
 1. A batch process for obtaining polynucleotidefragments having a selected size from a mixture of polynucleotidefragments comprising the steps of a) applying a solution of said mixtureof polynucleotide fragments and a counterion agent to a binding mediumhaving a hydrophobic surface; b) contacting the binding medium with afirst stripping solvent and counterion agent, the first strippingsolvent having a concentration of organic component sufficient torelease from the binding medium all polynucleotide fragments having asize smaller than the selected size, and removing the first strippingsolvent from the binding medium; and c) contacting the binding mediumwith a second stripping solvent having a concentration of organiccomponent sufficient to release from the binding medium thepolynucleotide fragments having the selected size, and removing thesecond stripping solvent from the binding medium.
 2. A process of claim1 wherein the binding medium is rinsed with fresh first strippingsolvent following step b) to remove residual fragments having a sizesmaller than the selected size therefrom.
 3. A process of claim 1wherein the binding medium is rinsed with fresh second stripping solventfollowing step c) to remove residual polynucleotide fragments of theselected size therefrom.
 4. A process of claim 1 wherein said counterionagent is selected from the group consisting of trialkylammonium acetate,trialkylammonium carbonate, and trialkylammonium phosphate.
 5. A processof claim 1 where said counterion agent is selected from the groupconsisting of triethylammonium acetate and triethylammoniumhexafluoroisopropyl alcohol.
 6. A process of claim 1 wherein saidorganic component is selected from the group consisting of alcohols,nitriles, dimethylformamide, esters, and ethers.
 7. A process of claim 6wherein said organic component comprises acetonitrile.
 8. A process ofclaim 1 wherein said binding medium comprises beads.
 9. A process ofclaim 8 wherein said beads are nonporous.
 10. A process of claim 8wherein said beads are porous.
 11. A process of claim 8 wherein saidbeads are comprised of organic polymer.
 12. A process of claim 8 whereinsaid beads are comprised of a copolymer of vinyl aromatic monomersselected from the group consisting of styrene, alkyl substitutedstyrene, alpha-methylstyrene and alpha substituted alpha-methylstyrene.13. A process of claim 8 wherein said beads have a diameter of fromabout 1.0 to 1,000 μm.
 14. A process of claim 8 wherein said beadscomprise inorganic particles selected from the group consisting ofsilica, silica carbide, silica nitrite, titanium oxide, aluminum oxide,zirconium oxide.
 15. A process of claim 14 wherein the hydrophobicsurface comprises an organic polymer supported on the inorganicparticle.
 16. A process of claim 15 wherein the hydrophobic surfaceincludes long chain hydrocarbons having from 8-24 carbons bonded to theinorganic particle.
 17. A process of claim 16 wherein any residual polargroups of the inorganic particle have been end-capped withtrimethylsilyl chloride or hexamethyldisilazane.
 18. A process of claim1 wherein said polynucleotides comprise double-stranded DNA.
 19. Aprocess of claim 1 wherein said mixture comprises the product of a PCRamplification.
 20. A process of claim 1 wherein said medium is containedwithin a column, a web or a container.
 21. A process of claim 1 whereinsaid medium is contained within a web said web comprising a compositearticle comprising: an inert fiber matrix and beads enmeshed in saidmatrix.
 22. A process of claim 21 wherein said web comprises: apolymeric fibril matrix and beads enmeshed in said matrix, the ratio ofbeads to fibril matrix being in the range of 29:1 to 4:1 by weight. 23.A process of claim 22 wherein said polymeric fibril ispolytetrafluroethylene.
 24. A process of claim 1 wherein said separationis by MIPC.